In Vitro Biosynthesis of the Core Scaffold of the Thiopeptide Thiomuracin

Abstract

Thiopeptides are potent antibiotics that inhibit protein synthesis. They are made by a remarkable post-translational modification process that transforms a linear peptide into a polycyclic structure. We present here the in vitro biosynthesis of the core scaffold of thiomuracin catalyzed by six proteins. We show that cyclodehydration precedes dehydration, and that dehydration is catalyzed by two proteins in a tRNAGlu-dependent manner. The enzyme that generates the pyridine core from two dehydroalanines ejects the leader peptide as a C-terminal carboxamide. Mutagenesis studies of the enzyme TbtD identified important residues for a formal [4+2] cycloaddition process. The core structure of thiomuracin exhibits similar antimicrobial activity to other known congeners, illustrating that in vitro biosynthesis is a viable route to potent antibiotics that can be explored for the rapid and renewable generation of analogues.

Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

“…S5). A similar pattern of multiple glutamylations has previously been observed for nonthiopeptide RiPP pathways (22,25); however, in the in vitro reconstituted thiomuracin system, only a monoglutamylated intermediate accumulates when the TclL ortholog (TbtC) is omitted (11). This discrepancy may be due to the different reaction conditions (in vitro versus in vivo) or to an intrinsic difference in how this two-step dehydration process is coordinated in the two biosynthetic systems.…”

“…Previous studies showed only MP1 is produced from the full set of genes, whereas only MP2 is formed in the ΔTclS strain (13). the recent in vitro thiomuracin study (11), may initiate processing. Indeed, the TclE peptide remains unmodified in the TclJ-defective strain (Fig.…”

“…These studies succeed in identifying extractable products and intermediates that have been prematurely cleaved from the leader peptide, but overlook peptide intermediates that are chemically very different from product molecules. In vitro studies of thiopeptide biosynthetic components have provided information about functional sets of enzymatic processing (11,12), but complete reconstitution remains a challenging endeavor. As an alternative approach, we recently reported an in vivo heterologous expression system that allows both efficient genetic changes in the core and biosynthetic enzymes and isolation and characterization of biochemical intermediates in micrococcin biosynthesis (13).…”

Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis

“…For a typical thiopeptide gene cluster in nature, one often encounters numerous additional genes that obscure the simplicity of central thiopeptide chemistry. The recent in vitro reconstitution of the thiomuracin biosynthetic pathway illustrates this simplicity (17). In that study, five different protein enzymes, along with an additional RiPP precursor peptide recognition element (RRE)-containing protein, were found to be sufficient for the synthesis of a bioactive thiopeptide scaffold.…”

“…In 2009, multiple independent researchers reported this general scheme for thiopeptide synthesis (11)(12)(13)(14)(15). In the years since, several additional biosynthetic principles have been elucidated (16)(17)(18). The process appears to involve three core steps: (i) cyclodehydration/oxidation of Cys residues to form thiazole heterocycles (Ser and Thr residues may also be modified to oxazoline or oxazole), (ii) dehydration of Ser and Thr residues yielding dehydroalanines and dehydrobutyrines, respectively, and (iii) cycloaddition of two of the dehydrated residues to yield the final macrocyclic thiopeptide scaffold (Fig.…”